1. Field
The present invention relates to novel use of leucyl tRNA synthetase and more particularly it relates a method of screening an agent for preventing or treating mTORC1 mediated diseases by screening an test agent which inhibits binding ability of LRS to RagD or RagD GTPases, and a method of reducing cell size as compared to the control group, comprising inhibiting expression of intracellular LRS.
2. Discussion of the Background
A leucine is one of three branched chain amino acids. Unlike other amino acids, leucine and the other branched chain amino acids, isoleucine and valine, escape liver metabolism due to the defect of the branched chain amino acid aminotransferase and directly influence muscle protein synthesis. Leucine not only serves as a substrate for protein synthesis but also is recognized as a potent signal nutrient that regulates protein metabolism. Oral administration of leucine increases rates of skeletal muscle protein synthesis in rats (Crozier S J, et. al., J Nutr. 135 (2005), 376382) and removal of leucine from a complete meal prevents stimulation of protein synthesis (Stipanuk M H., Nutr Rev. 65 (2007), 122129). Leucine-induced protein synthesis is mediated by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is composed of mTOR, regulatory associated protein of mammalian target of rapamycin (Raptor), G-protein βsubunit-like protein (GβL), and ras homolog enriched in brain (Rheb) (Bhaskar P T, et. al., Dev Cell. 12 (2007), 487502). mTORC1 phosphorylates S6K and 4E-BP, the rate-limiting step in translation, resulting in the translation initiation of mRNAs displaying a 5′ cap structure (Ma X M, Nat Rev Mol Cell Biol., 10(2009), 307-318; Holz M K, et. al., Cell 123(2005), pp 569-580).
mTORC1 regulates translation and cell growth by coordinating several upstream inputs such as growth factors, intracellular energy status, and amino acid availability. The Tuberous Sclerosis Complex (TSC) 1 and TSC2 regulate GTP/GDP exchange of Ras-like GTPase, Rheb to transmit growth factor and intracellular energy signals to mTORC1. When bound to GTP, Rheb interacts with and activates mTORC1 (Tee A R, et. al., Curr Biol. 13 (2003), 12591268) and appears to be necessary for the activation of mTORC1 by all signals, including amino acid availability. In contrast, TSC1-TSC2 is dispensable for the regulation of mTORC1 by amino acids, and, in cells lacking TSC2, the mTORC1 pathway is sensitive to amino acid starvation but resistant to growth factor withdrawal (Roccio M, et. al., Oncogene. 25 (2006), 657-664).
Recently, the Rag GTPases, which are also the members of the Ras family of GTP-binding proteins, were shown to be amino acid-specific regulators of the mTORC1 pathway (Sancak Y, et. al., Cell 141 (2010), 290-303). Mammals express four Rag proteins—RagA, RagB, RagC, and RagD—form heterodimers consisting of RagA or RagB with RagC or RagD. RagA and RagB, like RagC and RagD, are highly similar to each other and are functionally redundant (Schurmann A, et. al., J Biol Chem. 270 (1995), 28982-28988). Rag heterodimers containing GTP-bound RagB interact with mTORC1, and amino acids induce the mTORC1-Rag interaction by promoting the loading of RagB with GTP, which enables it to directly interact with the Raptor component of mTORC1 (Sancak Y, et. al., Cell 141 (2010), 290-303; Kim E, Nat Cell Biol. 10 (2008), 935-945). The activation of the mTORC1 pathway by amino acids correlates with the movement of mTORC1 from an undefined location to a compartment containing Rab7 (Sancak Y, et. al., Science 320 (2008), 1496-1501), a marker of both late endosomes and lysosomes (Bucci C, et. al., Mol Biol Cell., 11 (2000), 467-480). Recent report shows that amino acids induce the movement of mTORC1 to lysosome, where the Rag GTPases reside. Ragulator complex, which is composed of MAPKSP1, ROBLD3, and c11orf59 gene products, interacts with the Rag GTPases, recruits them to lysosomes and is essential for mTORC1 activation (Sancak Y, et. al., Cell 141 (2010), 290-303). However, how intracellular leucine is sensed for mTORC1 activation and how GTP/GDP cycles of Rag GTPases are regulated by amino acid for mTORC1 activation are unknown.
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for cellular protein synthesis and viability that catalyze the ligation of specific amino acids to their cognate tRNAs. The enzyme reaction is separated into two steps: the ATP-PPi exchange reaction for amino acid activation and aminoacylation of tRNA (Park S, et. al., Trends Biochem Sci. 30 (2005), 569-574). Based on amino acid sequence alignments and structural features, ARSs have been divided into two classes (Eriani G, et. al., Nature 347 (1990), 203-206; Burbaum J J, et. al., J Biol Chem. 266 (1991), 16965-16968). The class I synthetases share two consensus sequences, the HIGH (His-Ile-Gly-His) and KMSKS (Lys-Met-Ser-Lys-Ser) motifs, that form a nucleotide binding Rossmann fold (Arnez J G, et. al., Trends Biochem sci. 22 (1997), 211-216). In contrast, the class II synthetases do not contain the Rossmann fold, but share a very different catalytic domain (Cusack S, et. al., Nucl Acids Res. 19 (1991), 3489-3498). Leucyl-tRNA synthetase (LRS) is the class I enzyme, which is characterized by the HIGH and KMSKS motifs (Cusack S, et. al., EMBO J. 19 (2000), 2351-361). Structurally, LRS consists of the catalytic domain of bipartite Rossmann fold with a large insertion domain called CP1, a tRNA-binding anticodon domain, and a C-terminal extension domain (Cusack S, et. al., EMBO J. 19 (2000), 2351-361). In higher eukaryotic cells, LRS exists as a component of the ARS complex consisting of nine different tRNA synthetases and three non-enzymatic components, p18/AIMP3, p38/AIMP2, and p43/AIMP1 (Lee S W, et. al., J Cell Sci. 117 (2004), 3725-3734; Park S, et. al., Trends Biochem Sci. 30 (2005), 569-574; Park S G, et. al., Proc Natl Acad Sci USA 105 (2008), pp. 11043-11049). It has been shown that the C-terminal domain of LRS is crucial for the interaction with other components of the ARS complex (Ling C, et. al., J Biol Chem. 280 (2005), 34755-3463). Among the components of the complex, several different components are involved in various cell signaling processes (Lee Y N, et. al., Immunity 20 (2004), 145?51; Park S, et. al., Trends Biochem Sci. 30 (2005), pp. 569-574; Park S G, et. al., Proc Natl Acad Sci USA 105 (2008), pp. 11043-11049). For instance, glutamyl-prolyl-tRNA synthetase (EPRS) suppresses translation of the target inflammatory mRNAs by forming an interferon gamma-activated inhibitor of translation (GAIT) complex (Sampath P, et. al., Cell. 119 (2004), 195-208). Lysyl-tRNA synthetase (KRS) and its product, Ap4A, function as signaling regulators in the immune response by regulating gene expression (Lee Y N, et. al., Immunity 20 (2004), 145?51; Yannay-Cohen N, et. al., Mol Cell 34 (2009), 603-611). Methionyl-tRNA synthetase (MRS) and glutaminyl-tRNA synthetase (QRS) are involved in rRNA biogenesis (Ko et al., 2000) and anti-apoptotic signal regulation (Ko Y G, et. al., J Cell Biol 149 (2000), 567-574), respectively. Besides, cytosolic LRS was reported to be potentially implicated in lung cancer growth (Shin S H, et. al., Exp Mol Med. 40 (2008), 229-236), and the mitochondrial LRS may be involved in diabetes ('t Hart L M, et. al., Diabetes. 54 (2005), 1892-1895; Li R., et. al., Mol Cell Biol. 30 (2010), 2147-154).